Current knowledge as to the physiology of addiction designates the most influential target for drugs of abuse to be G-protein coupled receptors (GPCRs). GPCRs and the components associated with their signaling pathways are also important pharmaceutical targets. Yet, with very few exceptions, there is little direct structural information pertaining to these cell-surface receptors, and homology modeling has been widely used to gain provisional structural insight into most other GPCRs. The research proposed herein is directed at elucidating the structural components controlling the regulation and function of the human CB2 cannabinoid receptor (hCB2), a peripheral GPCR currently under intense investigation. Structural analyses of GPCRs by traditional methods such as x-ray crystallography have been hindered due to the arduous task of purifying adequate amounts of integral membrane protein in its intact native conformation. Consequently, I propose to pursue an alternative method in order to gain direct experimental information about GPCR tertiary structure by focusing primarily on the hCB2 receptor. For this purpose, specific cannabinergic ligands will be utilized (several as yet to be synthesized), as molecular probes directed to cysteine residues to identify and characterize hCB2's binding site(s) and to deduce receptor conformation(s) and structural features regarding activation and inactivation. In this strategy a reactive moiety will be strategically linked to a classical, high-affinity hCB2 cannabinoid agonist at distinct pharmacophoric positions. The reactive moiety will also be linked to a diarylpyrazole inverse-agonist congener. I propose to use these designer ligands as covalent affinity labels in tandem with expressed cysteine to serine or alanine site-directed hCB2 mutants to determine the ligands'site(s) of interaction and thereby deduce information regarding hCB2 receptor-ligand interaction (the cysteine knockouts will identify the region where the reactive moiety binds). Information gleaned from the empirical analyses of mutated and WT hCB2 challenged with the various cannabinergic ligands through binding and functional assays will be incorporated as constraints which may then be used to generate and refine three-dimensional computational models. Corroboration of experimental structural results may also be performed via mass spectroscopic analysis of covalently tagged, expressed receptor protein. PUBLIC HEALTH RELEVANCE: The strategies designed within this proposal will provide significant enhancement towards elucidating CB2 tertiary structure. A better understanding as to the regulation and function of GPCRs through ligand recognition and structural analysis will advance the scientific communities understanding of the molecular signals associated with drugs of abuse. This information will also aid in the design and synthesis of therapeutically relevant compounds.